Multi-target antimicrobial compositions comprising chitosan

ABSTRACT

The present application is directed to antimicrobial compositions comprising chitosan polymers of at least two different average molecular weights (Mws), wherein the Mw of each different chitosan polymer is selected to target a different microorganism as well as method of making and using the compositions and product comprising the compositions.

RELATED APPLICATIONS

The present application claims the benefit of priority from co-pendingU.S. Provisional Patent Application No. 62/555,722 filed on Sep. 8, 2017the contents of which are incorporated herein by reference.

FIELD

The present application is in the field of antimicrobial compositions,in particular natural antimicrobial compositions that target multiplemicroorganisms in the same product.

INTRODUCTION

The presence of microorganisms such as Gram positive and Gram negativebacteria, yeast and fungi, cause the spoilage of food, beverages,cosmetics, and personal care items, and lead to unsanitary medicaldevices and pharmaceuticals that poses serious risks for patients. Inaddition to causing health risks to the human consumer, spoilagerepresents wasted resources and energy, and lost revenue to producers.Preservatives reduce or eliminate microbial spoilage and are thus vitalingredients to a wide variety of products.

Natural preservative agents are growing in demand due to increasedconsumer awareness of the potential risks associated with artificialpreservative ingredients such as benzoates and sorbates. Consumer choiceincreasingly deviates from products listing artificial preservatives asan ingredient. Naturally derived mushroom chitosan can inhibit a broadrange of microorganisms and is well known to be a safe and naturalcompound. Chitosan from mushroom is especially useful for use as apreservative since it is non-animal, free of potential allergens, andcan be kosher and halal.

Chitosan, a deacetylated form of the biopolymer chitin, is a widelyabundant natural compound produced in the cell wall of, crustaceans,fungi, and insects. In the mushroom cell wall, chitin and chitosanprovide structure to the cell by maintaining its shape and providingstrength. Extraction of chitosan from mushroom biomass is relativelysimple and can be considered a “green” process due to the lowrequirement for energy, water and other chemicals. Mushroom derivedchitosan's high purity and non-animal origin present advantages for usein commercial applications such as in food and beverages, cosmetics,medical devices, and pharmaceuticals.

Mushroom chitosan is a dietary fiber that is non-toxic, highlybiocompatible, and has a long history of safe use in dietarysupplements, cosmetics, medical devices and pharmaceuticals.

Chitosan has been shown to be an effective antimicrobial against bothGram positive and Gram negative bacteria, yeasts and fungi. This broadrange of antimicrobial effectiveness is an improvement from some othernatural antimicrobials such as natamycin, which is only effectiveagainst yeast and fungi, and nisin, which is only effective against Grampositive bacteria.

The ability of chitosan to be an effective antimicrobial depends on itsmolecular properties including the percent degree of deacetylation (%DDA) and the molecular weight average (Mw). Generally the higher thepercent degree of deacetylation the more effective the chitosan isagainst all types of microorganisms. More important for theeffectiveness of chitosan as an antimicrobial is its Mw. Differentspecies of microorganism are impacted differently by different types ofchitosan polymer depending on the Mw of the chitosan polymer. Thus, somechitosan Mws are more effective than others at inhibiting the growth ofcertain types of microorganisms.

In most microbial contaminations, it is often a group of differentspecies of microorganisms producing a deleterious effect as opposed to asingle species. An ideal antimicrobial agent inhibits all of the harmfulspecies as opposed to just a single species.

SUMMARY

The present application includes antimicrobial compositions that usenaturally sourced preservatives and that are effective against multiplestrains of microbial species, and that are tailorable to differentmicrobial species combinations for different applications. The optimumMw of chitosan polymers to be used to inhibit the growth of differenttypes of microorganisms has been determined herein. Using thisknowledge, custom antimicrobial and/or preservative compositions havebeen developed to target or focus on specific applications based on theabundance of each type of spoilage microorganism present or expected tobe normally be present during the spoilage. Therefore compositions thatcontain the optimum ratio of chitosan polymer Mws for a specificproduct, for example, a specific food, beverage, cosmetic, personal careitem, medical device, or pharmaceutical, can be prepared.

In some embodiments, the present application includes an antimicrobialcomposition comprising chitosan polymers of at least two differentaverage molecular weights (Mws), wherein the Mw of each differentchitosan polymer is selected to target a different microorganism.

The application further includes a method of making the compositions ofthe application. In some embodiments, the method comprises:

a) obtaining specific chitosan polymers having different Mws determinedto be most effective at inhibiting growth of each target microorganism;andb) combining the specific chitosan polymers having different Mws inpercentage amounts based on the abundance of each type of microorganismpresent or expected to be present in a target product.

The present application further includes a product comprising one ormore compositions of the application.

Also included in the present application is a method of inhibitingmicrobial growth in a product comprising incorporating an effectiveamount of one or more of the compositions of the application into theproduct as well as a method of preserving a product comprisingincorporating an effective amount of one or more of the compositions ofthe application into the product.

Other features and advantages of the present application will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating embodiments of the application, are given byway of illustration only and the scope of the claims should not belimited by these embodiments, but should be given the broadestinterpretation consistent with the description as a whole.

DESCRIPTION OF VARIOUS EMBODIMENTS I. Definitions

Unless otherwise indicated, the definitions and embodiments described inthis and other sections are intended to be applicable to all embodimentsand aspects of the present application herein described for which theyare suitable as would be understood by a person skilled in the art.

The term “composition of the application” or “composition of the presentapplication” and the like as used herein refers to a compositioncomprising two or more different chitosan polymers, each chitosanpolymer having a different average molecular weight (Mw).

The term “average molecular weight” or “Mw” as used herein refers to theweight average molecular weight as measured in kiloDaltons (kDa) usingSize Exclusion Chromatography (SEC) or Gel Permeation Chromatography(GPC).

The term “and/or” as used herein means that the listed items arepresent, or used, individually or in combination. In effect, this termmeans that “at least one of” or “one or more” of the listed items isused or present.

As used in the present application, the singular forms “a”, “an” and“the” include plural references unless the content clearly dictatesotherwise. For example, an embodiment including “a microorganism” shouldbe understood to present certain aspects with one microorganism or twoor more additional microorganisms.

In embodiments comprising an “additional” or “second” component, such asan additional or second microorganism, the second component as usedherein is different from the other components or first component. A“third” component is different from the other, first, and secondcomponents, and further enumerated or “additional” components aresimilarly different.

In understanding the scope of the present application, the term“comprising” and its derivatives, as used herein, are intended to beopen ended terms that specify the presence of the stated features,elements, components, groups, integers, and/or steps, but do not excludethe presence of other unstated features, elements, components, groups,integers and/or steps. The foregoing also applies to words havingsimilar meanings such as the terms, “including”, “having” and theirderivatives.

The term “consisting” and its derivatives, as used herein, are intendedto be closed terms that specify the presence of the stated features,elements, components, groups, integers, and/or steps, but exclude thepresence of other unstated features, elements, components, groups,integers and/or steps.

The term “consisting essentially of”, as used herein, is intended tospecify the presence of the stated features, elements, components,groups, integers, and/or steps as well as those that do not materiallyaffect the basic and novel characteristic(s) of features, elements,components, groups, integers, and/or steps.

The term “suitable” as used herein means that the selection of theparticular compound or conditions would depend on the specific syntheticmanipulation to be performed, and the identity of the molecule(s) to betransformed, but the selection would be well within the skill of aperson trained in the art.

The terms “about”, “substantially” and “approximately” as used hereinmean a reasonable amount of deviation of the modified term such that theend result is not significantly changed. These terms of degree should beconstrued as including a deviation of at least ±5% of the modified termif this deviation would not negate the meaning of the word it modifiesor unless the context suggests otherwise to a person skilled in the art.

As used herein, the term “effective amount” means an amount of one ormore compositions of the application that is effective to achieve thedesired result. For example in the context of inhibiting microbialgrowth, an effective amount is an amount that, for example, increasessaid inhibition compared to the inhibition without administration of theone or more compositions.

II. Compositions of the Application

The present application includes an antimicrobial composition comprisingchitosan polymers of at least two different average molecular weights(Mws), wherein the Mw of each different chitosan polymer is selected totarget a different microorganism.

In some embodiments, the Mw of each different chitosan polymer isselected to focus on or target a different microorganism known to infecta specific product. In some embodiments, the specific product is aproduct that is susceptible to microbial growth. In some embodiments,the product is a food product, a beverage product, a cosmetic product, apersonal care item, a medical device or a pharmaceutical product.Pharmaceutical products include both prescription and over-the-counterpharmaceutical products. The product may be in any form, includingliquid and solid and combinations thereof.

In some embodiments, the microorganism target for the antimicrobialcompositions of the application determines how to select the amount andsize of each chitosan polymer to be included. Therefore if the targetmicroorganism is bacteria, the composition comprises substantiallygreater amounts in percent by weight of chitosan polymers having a Mw ofabout 30 kDa, about 40 kDa, about 150 kDa and/or about 170 kDa. If thebacteria are Gram positive, the composition comprises substantiallygreater amounts in percent by weight of chitosan polymers having a Mw ofabout 150 kDa and/or about 170 kDa. If the bacteria are Gram negative,the composition comprises substantially greater amounts in percent byweight of chitosan polymers having a Mw of about 30 kDa and/or about 40kDa. If the target microorganism is yeast, the composition comprisessubstantially greater amounts in percent by weight of chitosan polymershaving a Mw of about 20 kDa and/or about 60 kDa. If the targetmicroorganism is mold, the composition comprises substantially greateramounts in percent by weight of chitosan polymers having a Mw of about200 kDa.

If the target microorganisms include two of bacteria, mold and yeasts,then the antimicrobial composition comprises substantially greateramounts of chitosan polymers that target those organisms. Accordingly acomposition targeting bacteria and mold will comprise substantiallygreater amounts in percent by weight of chitosan polymers having a Mw ofabout 30 kDa, about 40 kDa, about 150 kDa and/or about 170 kDa andchitosan polymers having a Mw of about 200 kDa. A composition targetingbacteria and yeast will comprise substantially greater amounts inpercent by weight of chitosan polymers having a Mw of about 30 kDa,about 40 kDa, about 150 kDa and/or about 170 kDa and chitosan polymershaving a Mw of about 20 kDa and/or about 60 kDa. A composition targetingyeast and mold will comprise substantially greater amounts in percent byweight of chitosan polymers having a Mw of about 20 kDa and/or about 60kDa, and chitosan polymers having a Mw of about 200 kDa.

If the target microorganisms include all three of bacteria, mold andyeasts, then the antimicrobial composition may be a broad spectrumantimicrobial composition described in greater detail below. Thepercentages of each chitosan polymer will depend on which type ofmicroorganism one wishes to target more, which may depend on, forexample, a product's susceptibility to each microorganism. None-the-lessa person skilled in the art can determine the amounts and Mw of eachchitosan polymer based on the activity data disclosed herein andknowledge of the microorganisms to target.

In some embodiments, the term “substantially greater amounts in percentby weight” means that the referenced chitosan polymer is present in thecomposition in amounts that are about 5 percentage points (w/w) to about100 percentage points (w/w), or about 7 percentage points (w/w) to about50 percentage points (w/w), or about 10 percentage points (w/w) to about30 percentage points (w/w) greater than other chitosan polymers in thecomposition. For example, if the composition comprises 4 differentchitosan polymers, each having a different Mw, and one chitosan polymerwas present in about 20 percentage points greater than the other three,then the composition would comprise about 40% by weight of the polymerpresent in greater amounts and about 20% of each the remaining threepolymers.

In some embodiments, the antimicrobial composition is a broad spectrumcomposition and comprises at least 4 different chitosan polymers eachhaving a different Mw and each present in substantially equalpercentages by weight, wherein the 4 different chitosan polymers have aMw of:

(1) about 20 kDa or about 60 kDa;(2) about 30 kDa or about 40 kDa;(3) about 150 kDa or about 170 kDa; and(4) about 200 kDa.

In some embodiments, the broad spectrum antimicrobial compositioncomprises substantially equal percentages by weight of chitosan polymershaving a Mw of about 20 kDa, about 60 kDa, about 30, about 40 kDa, about150 kDa, about 170 kDa, and about 200 kDa.

In some embodiments, the broad spectrum antimicrobial compositiontargets yeasts and comprises substantially equal percentages by weightof chitosan polymers having a Mw of:

(1) about 30 kDa or about 40 kDa;(2) about 150 kDa or about 170 kDa; and(3) about 200 kDa; and(4) a substantially greater percentage by weight of a chitosan polymerhaving a Mw of about 20 kDa or about 60 kDa.

In some embodiments, the broad spectrum antimicrobial composition thattargets yeasts comprises substantially equal percentages by weight ofchitosan polymers having a Mw of about 30, about 40 kDa, about 150 kDa,about 170 kDa, and about 200 kDa, and a substantially greater percentageby weight of a chitosan polymer having a Mw of about 20 kDa and achitosan polymer having a Mw of about 60 kDa.

In some embodiments, the broad spectrum antimicrobial compositiontargets mold and comprises substantially equal percentages by weight ofchitosan polymers having a Mw of:

(1) about 20 kDa or about 60 kDa(2) about 30 kDa or about 40 kDa; and(3) about 150 kDa or about 170 kDa; and(4) a substantially greater percentage by weight of a chitosan polymerhaving a Mw of about 200 kDa.

In some embodiments, the broad spectrum antimicrobial composition thattargets mold comprises substantially equal percentages by weight ofchitosan polymers having a Mw of about 20 kDa, about 60 kDa, about 30,about 40 kDa, about 150 kDa, and about 170 kDa, and a substantiallygreater percentage by weight of a chitosan polymer having a Mw of about200 kDa.

In some embodiments, the broad spectrum antimicrobial compositiontargets bacteria and comprises substantially equal percentages by weightof chitosan polymers having a Mw of:

(1) about 20 kDa or about 60 kDa; and(2) about 200 kDa; and(3) a substantially greater percentage by weight of a chitosan polymerhaving a Mw of about 30 kDa or about 40 kDa; and(4) a substantially greater percentage by weight of a chitosan polymerhaving a Mw of about 150 kDa or about 170 kDa

In some embodiments, the broad spectrum antimicrobial composition thattargets bacteria comprises substantially equal percentages by weight ofchitosan polymers having a Mw of about 20, about 60 kDa and about 200kDa; and a substantially greater percentage by weight of a chitosanpolymer having a Mw of about 30 kDa and about 40 kDa, and a chitosanpolymer having a Mw of about 150 kDa and about 170 kDa.

In some embodiments, the broad spectrum antimicrobial compositiontargets Gram positive bacteria and comprises substantially equalpercentages by weight of chitosan polymers having a Mw of:

(1) about 20 kDa or about 60 kDa;(2) about 30 kDa or about 40 kDa; and(3) about 200 kDa; and(4) a substantially greater percentage by weight of a chitosan polymerhaving a Mw of about 150 kDa or about 170 kDa

In some embodiments, the broad spectrum antimicrobial composition thattargets Gram positive bacteria comprises substantially equal percentagesby weight of chitosan polymers having a Mw of about 20, about 60 kDa,about 30 kDa, about 40 kDa, and about 200 kDa; and a substantiallygreater percentage by weight of a chitosan polymer having a Mw of about150 kDa and about 170 kDa.

In some embodiments, the broad spectrum antimicrobial compositiontargets gram negative bacteria and comprises substantially equalpercentages by weight of chitosan polymers having a Mw of:

(1) about 20 kDa or about 60 kDa;(2) about 150 kDa or about 170 kDa; and(3) about 200 kDa; and(4) a substantially greater percentage by weight of a chitosan polymerhaving a Mw of about 30 kDa or about 40 kDa

In some embodiments, the broad spectrum antimicrobial composition thattargets Gram negative bacteria comprises substantially equal percentagesby weight of chitosan polymers having a Mw of about 20, about 60 kDa,about 150 kDa, about 170 kDa, and about 200 kDa; and a substantiallygreater percentage by weight of a chitosan polymer having a Mw of about30 kDa and about 40 kDa.

In some embodiments, the compositions comprise two or more chitosanpolymers that target two or more different types of Gram positivebacteria. For example, the antimicrobial composition comprises equalpercentages by weight of chitosan polymers having a Mw of about 150 kDaand about 170 kDa to target Lactobacillus and Listeria, respectively. Ifthe two target organisms are present in the product in unequal amountsthen the amount of each chitosan polymer is adjusted accordingly.

In some embodiments, the compositions comprise two or more chitosanpolymers that target two or more different types of Gram negativebacteria. For example, the antimicrobial composition comprises equalpercentages by weight of chitosan polymers having a Mw of about 30 kDaand about 40 kDa to target E. coli and Acetobacer, respectively. If thetwo target organisms are present in the product in unequal amounts thenthe amount of each chitosan polymer is adjusted accordingly.

In some embodiments, the compositions comprise two or more chitosanpolymers that target two or more different types of yeast. For example,the antimicrobial composition comprises equal percentages by weight ofchitosan polymers having a Mw of about 20 kDa and about 60 kDa to targetPichia and Saccharomyces, respectively. If the two target organisms arepresent in the product in unequal amounts then the amount of eachchitosan polymer is adjusted accordingly.

In some embodiments, the term “substantially equal percentages byweight” means that the referenced chitosan polymers are present in thecomposition in amounts that are the same within about +/−5% (wt/wt),about +/−4% (wt/wt), about +/−3% (wt/wt), about +/−2% (wt/wt) or about+/−1% (wt/wt).

In some embodiments, different Mw fractions of chitosan are isolatedusing chromatography, and/or by chemically, physically, or enzymaticallyaltering the molecular weight of a chitosan polymer isolated fromnatural sources to obtain the desired Mw fraction.

In some embodiments, the chitosan polymer is from a fungus, crustaceanor an insect. In some embodiments, the chitosan polymer is from afungus. In some embodiments, the chitosan polymer is from a mushroom.

In some embodiments, the compositions of the application are in the formof a powder. In some embodiments, the compositions of the applicationare in the form of a liquid solution or suspension. In some embodiments,the solvent for the solution or suspension is water. In someembodiments, the whole composition is dissolved into a solution.

In some embodiments, the compositions of the application include furtheringredients such as other preservative ingredients, other antioxidantingredients, colors, dyes, fillers and other excipients known in theart. In some embodiments, the compositions are formulated for inclusionin a specific product and ingredients are added to facilitate thisinclusion as would be known to a person skilled in the art.

III. Methods of Making Composition of the Application

The application further includes a method of making the compositions ofthe application. In some embodiments, the method comprises

a) obtaining specific chitosan polymers having different Mws determinedto be most effective at inhibiting growth of each target microorganism;andb) combining the specific chitosan polymers having different Mws inpercentage amounts based on the abundance of each type of microorganismpresent or expected to be present in a target product.

In some embodiments, the chitosan polymers having different Mws areobtained by dissolving chitosan from a natural source in weak acidicsolution, for example a solution of an organic acid such as acetic acidor lactic acid, and exposed for certain periods of time to a chemicalwhich will depolymerize the chitosan. In some embodiments, alkali basessuch as NaOH or KOH are used to chemically depolymerize the chitosansince the reaction occurs at a pace that allows the reaction to bestopped once the desired molecular weight has been achieved. Using analkali base also has another benefit of deacetylating the chitosansimultaneously while depolymerization occurs. In some embodiments,strong acids such as hydrochloric acid, or highly oxidizing chemicalslike hydrogen peroxide, are used to depolymerize chitosan quickly. Insome embodiments, the chitosan is depolymerized using enzymes. Thesemethods are used to isolate molecular weight fractions either the samesize or smaller than the largest molecular weight present in the naturalchitosan. For example, if the original natural chitosan is 200 kDa theisolated fractions are equal to or smaller than 200 kDa.

In some embodiments the chitosan polymers having different Mws areobtained by dissolving chitosan from a natural source in weak acidicsolution, for example a solution of an organic acid such as acetic acidor lactic acid. In some embodiments the chitosan is dissolved in theacidic solution in amounts to allow the solution to be fluid enough tobe pumped into a chromatography column. The different Mw fractions ofchitosan are then isolated from the column. In some embodiments, thedifferent Mw fractions of chitosan are isolated using other physicalmethods. In some embodiments, the isolated fractions of different Mwsare precipitated by adjusting the pH of a solution of each fractionto >7 using a base, such as sodium or potassium hydroxide (NaOH or KOH).In some embodiments, the precipitated chitosan is collected byfiltration or centrifugation and dried into a powder.

In some embodiments, the PDI of the chitosan Mw fractions is about 2 toabout 3.

In some embodiments, molecular weights are analyzed by HPLC with sizeexclusion chromatography or gel permeation chromatography.

In some embodiments, the different Mw polymers of chitosan are combinedas powders. In some embodiments, the different Mw polymers of chitosanare combined as solutions or are combined in a solution or suspension.

Obtaining chitosan polymer fractions having a desired Mw has certainchallenges. Variables such as reaction time, temperature and agitationspeed, and starting Mw have to be taken into consideration as each willaffect the size and PDI of the resulting product. Further, there can becontinued depolymerization during precipitation and collection stepswhich must be taken into consideration. The present Applicant hasperfected this method to isolate chitosan fractions having specificdesired Mw's and PDIs. To the best of the Applicant's knowledge no suchmethods existed previously therefore identification of the antimicrobialactivity for such a wide variety of chitosan Mws has not been possibleuntil now.

IV. Methods of Using and Product Containing Compositions of theApplication

In some embodiments, the compositions of the application are used asantimicrobial agents or preservatives in products in need of suchagents. In some embodiments, these products include, but are not limitedto food, beverages, cosmetics, personal care items, medical devices andpharmaceuticals. As noted above, pharmaceuticals include prescriptionand over-the-counter pharmaceuticals. The product may be in any form,including liquid, semi-solid and solid and combinations thereof.

Accordingly, the present application further includes a productcomprising one or more compositions of the application. In someembodiments, the product is a food, beverage, cosmetic, personal careitem, medical device or pharmaceutical.

In some embodiments, the compositions of the application areincorporated as an ingredient or a component during the preparation ofthe product.

In some embodiments, the application includes a method of inhibitingmicrobial growth in a product comprising incorporating an effectiveamount of one or more of the compositions of the application into theproduct.

In some embodiments, the application includes a method of preserving aproduct comprising incorporating an effective amount of one or more ofthe compositions of the application into the product.

In some embodiments, the application includes a use of one or more ofthe compositions of the application to inhibit microbial growth in aproduct or to preserve a product.

In some embodiments, the effective amount of the composition of theapplication will depend on the identity of the product. For example, ifthe product is a food or beverage product an effective amount may be anamount of the composition to deliver or incorporate about 10 ppm toabout 500 ppm, about 20 ppm to about 400 ppm, about 30 ppm to about 300ppm, about 40 ppm to about 250 ppm, or about 50 ppm to about 200 ppm ofthe chitosan polymers of at least two different average molecularweights (Mws) to the product. In a further example, if the product is acosmetic or personal care product an effective amount may be an amountof the composition to deliver or incorporate about 50 ppm to about 1000ppm, about 100 ppm to about 900 ppm, about 200 ppm to about 700 ppm,about 300 ppm to about 600 ppm, or about 400 ppm to about 500 ppm of thechitosan polymers of at least two different average molecular weights(Mws) to the product.

V. Examples Example 1: Preparation of Chitosan Polymers Having DifferentMWS

The chitosan molecular weight fractions were separated by chemicaldepolymerization using a sodium hydroxide solution. 10 g of chitosan wasdissolved in 250 mL of 1% (v/v) glacial acetic acid solution at 35° C.for 5 hours. 10 mL of 0.3M sodium hydroxide was added into the chitosansolution and the solution heated to 60° C. while being stirred with amagnetic stir bar. The starting Mw was measured using GPC according tothe method outlined below and depending on the starting Mw measurementthe chitosan was exposed for varying amounts of time to achieve thedesired Mw. Once the sufficient period of time has elapsed the reactionwas stopped by adding sodium hydroxide to raise the pH above 7 in orderto precipitate the chitosan and the precipitate was collected usingcentrifugation, rinsed with water, and dried using lyophilization. Thisprocess was repeated until each desired Mw was obtained. A general trendfor the rate of the reaction was that there was a rapid period ofdepolymerization where the Mw decreased from approximately 200 kDa toapproximately 100 kDa in 1 hour, and then a slow period where themolecular weight decreased from approximately 100 kDa to 10 kDa in over12 hours. In this example 21 different molecular weights were produced;3, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,160, 170, 180, 190 and 200 kDa.

The chitosan Mw fractions were analyzed by HPLC with size exclusionchromatography or gel permeation chromatography, and incorporating anoven that has four detectors: refractive index (RI), ultra-violet (UV),right angle and low angle light scattering (RALS/LALS), andfour-capillary differential viscometer (VIS). Chitosan samples weredissolved in the mobile phase of 0.1M acetic acid/0.3M NaNO₃ for 4-6hours on a rocker at room temperature. A concentration of chitosan at˜5-10 mg/mL was used for the samples. Columns can be used in a seriesand with a large range of separation efficiency. The isolated chitosanMw's used for antimicrobial testing had a PDI between 2 and 3.

Example 4: Broad Spectrum of Antimicrobial Screening of ChitosanFractions Having Different Mws

The microorganisms Listeria innocua (Seeliger ATCC 33090), Escherichiacoli (Migula ATCC 11229), Saccharomyces cerevisiae, Acetobacter aceti(Pasteur ATCC 15973), Lactobacillus casei (Orla-Jensen ATCC 393), Pichiaanomala (Hansen), and Rhizopus oryzae were used for this experiment. Themicroorganisms were kept frozen at −4° C. in BHI, Luria broth and PDBcontaining glycerol (10% v/v). Before use, the stock cultures wereresuscitated through 2 consecutive 24 h growth in Brain Heart Infusion(BHI) or Luria-Bertaini (LB) or Potato Dextrose Broth (PDB) at 37° C. toobtain the working cultures containing approximately 10⁹ CFU/mL.

The antimicrobial activity of different molecular weights of chitosan orformulations thereof were tested in vitro according to an agar diffusionassay against Listeria innocua, Escherichia coli, Saccharomycescerevisiae, Acetobacter aceti, Lactobacillus casei, Pichia anomala, andRhizopus oryzae. Test mediums containing BHI/LB/PDB and agar wereprepared, autoclaved and allowed to cool at 50° C. in a water bath. Testmediums BHI, LB and PDB were prepared into solution prior to beingautoclaved. The microorganisms were grown in BHI, LB, PDB and placed inan incubator at 37 (for bacteria) or 30° C. (for yeast and fungi) for 24or 48 hr to get the final microbial concentration 10⁹ CFU/mL. The mediumwas inoculated with 1.0% (v/v) BHI or LB or PDB containing the microbialcultures (10⁹ CFU/mL) to obtain a final microbial population ofapproximately 10⁶ CFU/mL. Then 1 mL of the inoculated medium was spreadto each Petri dish (95 mm×15 mm). Thereafter, holes measuring 7.0 mm indiameter were dug and 80-100 μL of the antimicrobial formulations atconcentration range of 10-500 ppm was added to each hole. The plateswere incubated for 24 or 48 h at 37 or 30° C. for bacteria andyeast/fungi respectively. The diameter (mm) of the inhibition zones (IZ)was measured to determine the antimicrobial activity of theformulations.

The effect of fungal chitosan samples having different Mws on the growthof a variety of microorganisms was assessed. In particular, the growthof various representative microbial organisms was observed in thepresence of fungal chitosan samples having molecular weights of 3, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, and 200 kDa each with a Polydispersity Index of between 2 and3. Each Mw sample was tested at a concentration of 10, 50, 100, 300, and500 ppm. The Mw of the chitosan sample showing the optimal inhibition ofmicrobial growth (by providing the largest growth inhibition zone (IZ)at 500 ppm) was observed and the results are summarized in Table 1.

TABLE 1 Microorganism Optimal IZ at Type Species Mw (kDa) 500 ppm (mm)Bacteria G− Escherichia coli 30 12 G− Acetobacter aceti 40 11.7 G+Lactobacillus casei 150 10.2 G+ Listeria innocua 170 12.7 Yeast Pichiaanomala 20 10.5 Yeast Saccharomyces cerevisiae 60 14.3 Mold Rhizopusoryzae 200 15

Example 5: Antimicrobial Compositions

Based on the above results, the following antimicrobial formulations areprepared:

(a) Broad spectrum anti-microbial chitosan composition

Mw % (w/w) 20 14.29 30 14.29 40 14.29 60 14.29 150 14.29 170 14.29 20014.29 100.00(b) Broad spectrum anti-microbial chitosan composition with a majorfocus on Yeast

Mw % (w/w) 20 30.00 30 8.00 40 8.00 60 30.00 150 8.00 170 8.00 200 8.00100.00(c) Broad spectrum anti-microbial chitosan composition with a minorfocus on Yeast

Mw % (w/w) 20 20.00 30 12.00 40 12.00 60 20.00 150 12.00 170 12.00 20012.00 100.00(d) Broad spectrum anti-microbial chitosan composition with a majorfocus on Mold

Mw % (w/w) 20 6.67 30 6.67 40 6.67 60 6.67 150 6.67 170 6.67 200 60.00100.00(e) Broad spectrum anti-microbial chitosan composition with a minorfocus on Mold

Mw % (w/w) 20 11.67 30 11.67 40 11.67 60 11.67 150 11.67 170 11.67 20030.00 100.00(f) Broad spectrum anti-microbial chitosan composition with a majorfocus on Bacteria

Mw % (w/w) 20 6.67 30 20.00 40 20.00 60 6.67 150 20.00 170 20.00 2006.67 100.00(g) Broad spectrum anti-microbial chitosan composition with a minorfocus on Gram + bacteria

Mw % (w/w) 20 12.00 30 12.00 40 12.00 60 12.00 150 20.00 170 20.00 20012.00 100.00(h) Broad spectrum anti-microbial chitosan composition with a majorfocus on Gram − bacteria

Mw % (w/w) 20 8.00 30 30.00 40 30.00 60 8.00 150 8.00 170 8.00 200 8.00100.00(i) Broad spectrum anti-microbial chitosan composition with a minorfocus on Gram − bacteria

Mw % (w/w) 20 12.00 30 20.00 40 20.00 60 12.00 150 12.00 170 12.00 20012.00 100.00(j) Broad spectrum anti-microbial chitosan composition with a majorfocus on Yeast and Mold

Mw % (w/w) 20 25.00 30 6.25 40 6.25 60 25.00 150 6.25 170 6.25 200 25.00100.00(k) Broad spectrum anti-microbial chitosan composition with a minorfocus on Yeast and Mold

Mw % (w/w) 20 20.00 30 10.00 40 10.00 60 20.00 150 10.00 170 10.00 20020.00 100.00(l) Broad spectrum anti-microbial chitosan composition with a majorfocus on Yeast and Mold and Gram + bacteria

Mw % (w/w) 20 18.00 30 5.00 40 5.00 60 18.00 150 18.00 170 18.00 20018.00 100.00(m) Broad spectrum anti-microbial chitosan composition with a minorfocus on Yeast and Mold and Gram + bacteria

Mw % (w/w) 20 15.00 30 12.50 40 12.50 60 15.00 150 15.00 170 15.00 20015.00 100.00(n) Broad spectrum anti-microbial chitosan composition with a majorfocus on Yeast and Mold and Gram− bacteria

Mw % (w/w) 20 18.00 30 18.00 40 18.00 60 18.00 150 5.00 170 5.00 20018.00 100.00(o) Broad spectrum anti-microbial chitosan composition with a minorfocus on Yeast and Mold and Gram− bacteria

Mw % (w/w) 20 15.00 30 15.00 40 15.00 60 15.00 150 12.50 170 12.50 20015.00 100.00(p) Anti-microbial chitosan composition with a focus on Yeasts

Mw % (w/w) 20 50.00 60 50.00 100.00(q) Antimicrobial chitosan composition for Yeasts with focus onSaccharomyces

Mw % (w/w) 20 25.00 60 75.00 100.00(r) Antimicrobial chitosan composition for Yeasts with focus on Pichia

Mw % (w/w) 20 75.00 60 25.00 100.00(s) Antimicrobial chitosan composition for Yeast and Molds

Mw % (w/w) 20 33.33 60 33.33 200 33.33 100.00(t) Antimicrobial chitosan composition for Yeast and Molds with focus onyeasts

Mw % (w/w) 20 40.00 60 40.00 200 20.00 100.00(u) Antimicrobial chitosan composition for Yeast and Molds with focus onmolds

Mw % (w/w) 20 25.00 60 25.00 200 50.00 100.00(v) Antimicrobial chitosan composition for Bacteria (Gram +& −)

Mw % (w/w) 30 25.00 40 25.00 150 25.00 170 25.00 100.00(w) Antimicrobial chitosan composition for Bacteria with a focus on Gram+

Mw % (w/w) 30 10.00 40 10.00 150 40.00 170 40.00 100.00(x) Antimicrobial chitosan composition for Bacteria with focus on Gram −

Mw % (w/w) 30 40.00 40 40.00 150 10.00 170 10.00 100.00(y) Antimicrobial chitosan composition for Yeast and Molds and Gram +bacteria

Mw % (w/w) 20 20.00 60 20.00 200 20.00 150 20.00 170 20.00 100.00(z) Antimicrobial chitosan composition for Yeast and Molds and Gram +bacteria with a focus on Yeasts

Mw % (w/w) 20 35.00 60 35.00 200 10.00 150 10.00 170 10.00 100.00(aa) Antimicrobial chitosan composition for Yeast and Molds and Gram +bacteria with a focus on Yeasts and Molds

Mw % (w/w) 20 25.00 60 25.00 200 25.00 150 12.50 170 12.50 100.00(bb) Antimicrobial chitosan composition for Yeast and Molds and Gram +bacteria with a focus on Molds

Mw % (w/w) 20 10.00 60 10.00 200 60.00 150 10.00 170 10.00 100.00(cc) Antimicrobial chitosan composition for Yeast and Molds and Gram +bacteria with a focus on Gram +

Mw % (w/w) 20 13.33 60 13.33 200 13.33 150 30.00 170 30.00 100.00(dd) Antimicrobial chitosan composition for Yeast and Molds and Gram +bacteria with a focus on Saccharomyces

Mw % (w/w) 20 12.50 60 50.00 200 12.50 150 12.50 170 12.50 100.00(ee) Antimicrobial chitosan composition for Yeast and Bacteria

Mw % (w/w) 20 25.00 60 25.00 30 12.50 40 12.50 150 12.50 170 12.50100.00(ff) Antimicrobial chitosan composition for Yeast and Bacteria with afocus on Yeast

Mw % (w/w) 20 25.00 60 25.00 30 12.50 40 12.50 150 12.50 170 12.50100.00(gg) Antimicrobial chitosan composition for Yeast and Bacteria with afocus on Bacteria

Mw % (w/w) 20 10.00 60 10.00 30 20.00 40 20.00 150 20.00 170 20.00100.00(hh) Antimicrobial chitosan composition for Yeast and Bacteria with afocus on Gram + bacteria

Mw % (w/w) 20 7.50 60 7.50 30 7.50 40 7.50 150 35.00 170 35.00 100.00(ii) Antimicrobial chitosan composition for Yeast and Bacteria with afocus on Gram − bacteria

Mw % (w/w) 20 7.50 60 7.50 30 35.00 40 35.00 150 7.50 170 7.50 100.00(jj) Antimicrobial chitosan composition for Yeast and Bacteria with afocus on Gram − bacteria and yeast

Mw % (w/w) 20 20.00 60 20.00 30 20.00 40 20.00 150 10.00 170 10.00100.00(kk) Antimicrobial chitosan composition for Yeast and Bacteria with afocus on Gram + bacteria and yeast

Mw % (w/w) 20 20.00 60 20.00 30 10.00 40 10.00 150 20.00 170 20.00100.00(ll) Antimicrobial chitosan composition for Yeast and Bacteria with afocus on Gram − bacteria and yeast

Mw % (w/w) 20 20.00 60 20.00 30 20.00 40 20.00 150 10.00 170 10.00100.00(mm) Antimicrobial chitosan composition for Yeast and Molds and Gram −bacteria

Mw % (w/w) 30 20.00 40 20.00 20 20.00 60 20.00 200 20.00 100.00(nn) Antimicrobial chitosan composition for Yeast and Molds and Gram −bacteria with a focus on Yeasts

Mw % (w/w) 30 35.00 40 35.00 20 10.00 60 10.00 200 10.00 100.00(oo) Antimicrobial chitosan composition for Yeast and Molds and Gram −bacteria with a focus on Yeasts and Molds

Mw % (w/w) 30 12.50 40 12.50 20 25.00 60 25.00 200 25.00 100.00(pp) Antimicrobial chitosan composition for Yeast and Molds and Gram −bacteria with a focus on Molds

Mw % (w/w) 30 10.00 40 10.00 20 10.00 60 10.00 200 60.00 100.00(qq) Antimicrobial chitosan composition for Yeast and Molds and Gram −bacteria with a focus on Gram −

Mw % (w/w) 30 30.00 40 30.00 20 13.33 60 13.33 200 13.33 100.00(rr) Antimicrobial chitosan composition for Yeast and Molds and Gram −bacteria with a focus on Saccharomyces

Mw % (w/w) 30 12.50 40 12.50 20 12.50 60 50.00 200 12.50 100.00(ss) Antimicrobial chitosan composition for Gram + bacteria

Mw % (w/w) 150 50.00 170 50.00 100.00(tt) Antimicrobial chitosan composition for Gram − bacteria

Mw % (w/w) 30 50.00 40 50.00 100.00

1. An antimicrobial composition comprising chitosan polymers of at leasttwo different average molecular weights (Mws), wherein the Mw of eachdifferent chitosan polymer is selected to target a differentmicroorganism.
 2. The antimicrobial composition of claim 1, wherein theMw of each different chitosan polymer is selected to target a differentmicroorganism known to infect a specific product.
 3. The antimicrobialcomposition of claim 2, wherein the target microorganism is bacteria andthe composition comprises substantially greater amounts in percent byweight of chitosan polymers having a Mw of about 30 kDa, about 40 kDa,about 150 kDa and/or about 170 kDa.
 4. The antimicrobial composition ofclaim 3, wherein the bacteria are Gram positive and the compositioncomprises substantially greater amounts in percent by weight of chitosanpolymers having a Mw of about 150 kDa and/or about 170 kDa.
 5. Theantimicrobial composition of claim 3, wherein the bacteria are Gramnegative and the composition comprises substantially greater amounts inpercent by weight of chitosan polymers having a Mw of about 30 kDaand/or about 40 kDa.
 6. The antimicrobial composition of claim 2,wherein the target microorganism is yeast and the composition comprisessubstantially greater amounts in percent by weight of chitosan polymershaving a Mw of about 20 kDa and/or about 60 kDa.
 7. The antimicrobialcomposition of claim 2, wherein the target microorganism is mold and thecomposition comprises substantially greater amounts in percent by weightof chitosan polymers having a Mw of about 200 kDa.
 8. The antimicrobialcomposition of claim 2, wherein the composition targets bacteria andmold and comprises substantially greater amounts in percent by weight ofchitosan polymers having a Mw of about 30 kDa, about 40 kDa, about 150kDa and/or about 170 kDa and chitosan polymers having a Mw of about 200kDa.
 9. The antimicrobial composition of claim 2, wherein thecomposition targets bacteria and yeast and comprises substantiallygreater amounts in percent by weight of chitosan polymers having a Mw ofabout 30 kDa, about 40 kDa, about 150 kDa and/or about 170 kDa andchitosan polymers having a Mw of about 20 kDa and/or about 60 kDa. 10.The antimicrobial composition of claim 2, wherein the compositiontargets yeast and mold and comprises substantially greater amounts inpercent by weight of chitosan polymers having a Mw of about 20 kDaand/or about 60 kDa, and chitosan polymers having a Mw of about 200 kDa.11. The antimicrobial composition of claim 1, wherein the composition isa broad spectrum composition and comprises at least 4 different chitosanpolymers each having a different Mw and each present in substantiallyequal percentages by weight, wherein the 4 different chitosan polymershave a Mw of: (1) about 20 kDa or about 60 kDa; (2) about 30 kDa orabout 40 kDa; (3) about 150 kDa or about 170 kDa; and (4) about 200 kDa.12. The antimicrobial composition of claim 11, wherein the broadspectrum antimicrobial composition comprises substantially equalpercentages by weight of chitosan polymers having a Mw of about 20 kDa,about 60 kDa, about 30, about 40 kDa, about 150 kDa, about 170 kDa, andabout 200 kDa.
 13. The antimicrobial composition of claim 1, wherein thecomposition is a broad spectrum composition that targets yeasts andcomprises substantially equal percentages by weight of chitosan polymershaving a Mw of: (1) about 30 kDa or about 40 kDa; (2) about 150 kDa orabout 170 kDa; and (3) about 200 kDa; and (4) a substantially greaterpercentage by weight of a chitosan polymer having a Mw of about 20 kDaor about 60 kDa.
 14. The antimicrobial composition of claim 13, whereinthe composition comprises substantially equal percentages by weight ofchitosan polymers having a Mw of about 30, about 40 kDa, about 150 kDa,about 170 kDa, and about 200 kDa, and a substantially greater percentageby weight of a chitosan polymer having a Mw of about 20 kDa and achitosan polymer having a Mw of about 60 kDa.
 15. The antimicrobialcomposition of claim 1, wherein the composition is a broad spectrumantimicrobial composition that targets mold and comprises substantiallyequal percentages by weight of chitosan polymers having a Mw of: (1)about 20 kDa or about 60 kDa (2) about 30 kDa or about 40 kDa; and (3)about 150 kDa or about 170 kDa; and (4) a substantially greaterpercentage by weight of a chitosan polymer having a Mw of about 200 kDa.16. The antimicrobial composition of claim 15, wherein the compositioncomprises substantially equal percentages by weight of chitosan polymershaving a Mw of about 20 kDa, about 60 kDa, about 30, about 40 kDa, about150 kDa, and about 170 kDa, and a substantially greater percentage byweight of a chitosan polymer having a Mw of about 200 kDa.
 17. Theantimicrobial composition of claim 1, wherein the composition is a broadspectrum antimicrobial composition that targets bacteria and comprisessubstantially equal percentages by weight of chitosan polymers having aMw of: (1) about 20 kDa or about 60 kDa; and (2) about 200 kDa; and (3)a substantially greater percentage by weight of a chitosan polymerhaving a Mw of about 30 kDa or about 40 kDa; and (4) a substantiallygreater percentage by weight of a chitosan polymer having a of about 150kDa or about 170 kDa
 18. The antimicrobial composition of claim 17,wherein the composition comprises substantially equal percentages byweight of chitosan polymers having a Mw of about 20, about 60 kDa andabout 200 kDa; and a substantially greater percentage by weight of achitosan polymer having a Mw of about 30 kDa and about 40 kDa, and achitosan polymer having a Mw of about 150 kDa and about 170 kDa.
 19. Theantimicrobial composition of claim 1, wherein the composition is a broadspectrum antimicrobial composition that targets Gram positive bacteriaand comprises substantially equal percentages by weight of chitosanpolymers having a Mw of: (1) about 20 kDa or about 60 kDa; (2) about 30kDa or about 40 kDa; and (3) about 200 kDa; and (4) a substantiallygreater percentage by weight of a chitosan polymer having a Mw of about150 kDa or about 170 kDa
 20. The antimicrobial composition of claim 19,wherein the composition comprises substantially equal percentages byweight of chitosan polymers having a Mw of about 20, about 60 kDa, about30 kDa, about 40 kDa, and about 200 kDa; and a substantially greaterpercentage by weight of a chitosan polymer having a Mw of about 150 kDaand about 170 kDa.
 21. The antimicrobial composition of claim 1, whereinthe composition is a broad spectrum antimicrobial composition thattargets Gram negative bacteria and comprises substantially equalpercentages by weight of chitosan polymers having a Mw of: (1) about 20kDa or about 60 kDa; (2) about 150 kDa or about 170 kDa; and (3) about200 kDa; and (4) a substantially greater percentage by weight of achitosan polymer having a Mw of about 30 kDa or about 40 kDa
 22. Theantimicrobial composition of claim 21, wherein the composition comprisessubstantially equal percentages by weight of chitosan polymers having aMw of about 20, about 60 kDa, about 150 kDa, about 170 kDa, and about200 kDa; and a substantially greater percentage by weight of a chitosanpolymer having a Mw of about 30 kDa and about 40 kDa.
 23. Theantimicrobial composition of any one of claims 3 to 22, whereinsubstantially greater amounts in percent by weight means that areferenced chitosan polymer is present in the composition in amountsthat are about 5 percentage points (w/w) to about 100 percentage points(w/w), or about 7 percentage points (w/w) to about 50 percentage points(w/w), or about 10 percentage points (w/w) to about 30 percentage points(w/w) greater than other chitosan polymers in the composition.
 24. Theantimicrobial composition of any one of claims 3 to 23, whereindifferent Mw fractions of chitosan are isolated using chromatography, orother physical methods, or by chemically or enzymatically altering themolecular weight of a chitosan polymer isolated from natural sources tothe desired fraction.
 25. The antimicrobial composition of any one ofclaims 3 to 23, wherein the chitosan polymer is from a fungus,crustacean or an insect.
 26. The antimicrobial composition of claim 25,wherein the chitosan polymer is from a fungus.
 27. The antimicrobialcomposition of claim 26, wherein the chitosan polymer is from amushroom.
 28. The antimicrobial composition of any one of claims 3 to27, wherein compositions are in the form of a powder, liquid solution orsuspension.
 29. The antimicrobial composition of claim 28, whereincompositions are in the form of a powder.
 30. The antimicrobialcomposition of any one of claims 3 to 27, wherein the whole compositionis dissolved into a solution.
 31. The antimicrobial composition of anyone of claims 3 to 30, wherein the compositions of the applicationinclude further ingredients such as other preservative ingredients,other antioxidant ingredients, colors, dyes, fillers and otherexcipients.
 32. The antimicrobial composition of any one of claims 3 to31, wherein the compositions are formulated for inclusion in a specificproduct.
 33. A method of making a composition of any one of claims 1 to32 comprising: a) obtaining specific chitosan polymers having differentMws determined to be most effective at inhibiting growth of each targetmicroorganism; and b) combining the specific chitosan polymers havingdifferent Mws in percentage amounts based on the abundance of each typeof microorganism present or expected to be present in a target product.34. The method of claim 33, wherein the different Mw polymers ofchitosan are combined as powders.
 35. A product comprising one or morecompositions of any one of claims 1 to
 32. 36. The product of claim 35,wherein the product is a food, beverage, cosmetic, personal care item,medical device or pharmaceutical.
 37. The product of claim 35 or 36,wherein the compositions are incorporated as an ingredient or acomponent during preparation of the product.
 38. A method of inhibitingmicrobial growth in a product comprising incorporating an effectiveamount of one or more of the compositions of any one of claims 1 to 32into the product.
 39. A method of preserving a product comprisingincorporating an effective amount of one or more of the compositions ofany one of claims 1 to 32 into the product.
 40. The method of claim 38or 39, wherein the product is a food, beverage, cosmetic, personal careitem, medical device or pharmaceutical.
 41. The method of claim 38 or39, wherein the product is a food or beverage product and the effectiveamount is an amount of the composition to deliver or incorporate about10 ppm to about 500 ppm, about 20 ppm to about 400 ppm, about 30 ppm toabout 300 ppm, about 40 ppm to about 250 ppm, or about 50 ppm to about200 ppm of the chitosan polymers of at least two different averagemolecular weights (Mws) to the product.
 42. The method of claim 38 or39, wherein the product is a cosmetic or personal care product and theeffective amount is an amount of the composition to deliver orincorporate about 50 ppm to about 1000 ppm, about 100 ppm to about 900ppm, about 200 ppm to about 700 ppm, about 300 ppm to about 600 ppm, orabout 400 ppm to about 500 ppm of the chitosan polymers of at least twodifferent average molecular weights (Mws) to the product.